Background: Adiposity in youth is inversely related to breast cancer risk throughout life, but the underlying mechanism is not understood. Breast density is one of the strongest breast cancer risk factors. In our ongoing research, adiposity in youth is strongly and significantly inversely associated with adult percent breast density as a consequence of heavier girls having less dense breast fibroglandular tissue. We also found youth saturated fat intake to be positively associated and unsaturated fat intake inversely associated with adult breast density. Objectives: The goal of this study is to identify physiological mechanisms that could mediate the associations of youth adiposity and dietary fat intake with breast density so targeted interventions can be developed. Specific Aims: Primary aims are to 1) identify metabolomic profiles in serum collected in youth associated with youth adiposity and young adult breast density; and 2) identify metabolomic profiles in serum collected in youth associated with youth fat subtype (saturated, polyunsaturated and monounsaturated fat) consumption and young adult breast density A secondary aim will formally evaluate mediation of associations of youth adiposity and dietary fat intake with breast density phenotypes by metabolites in serum collected in youth identified in aims 1 and 2. Study Design: We will use resources previously collected in the Dietary Intervention Study in Children (DISC) and DISC06 Follow-Up Study. DISC evaluated the safety and efficacy of a diet intervention to reduce LDL-cholesterol in children. 301 healthy girls 7-10 years old were enrolled in DISC and continued on study until they averaged 17 years old. Anthropometry was measured annually and dietary recalls and blood were collected biannually. DISC06 was conducted when participants were 25-29 years old to evaluate the longer-term effects of the intervention on biomarkers associated with breast cancer and included blood collection and assessment of anthropometry, diet and breast density. Approximately 300 participants will be included in analyses of associations of youth adiposity and diet with metabolites in serum collected during youth in DISC, while 182 participants will be included in analysis of associations with breast density phenotypes. Metabolomics profiles will be measured in stored serum collected during adolescence in DISC. Mixed effects linear regression will be used to identify individual metabolites associated with youth adiposity and dietary fat intake and adult breast density, while enrichment and pathway analysis will be used to identify relevant metabolic pathways. Significance: We are uniquely positioned to address an important and timely question that is highly innovative. Because it is a time of rapid proliferation of the mammary glands, adolescence may be a particularly vulnerable time for exposures related to breast cancer. Study results will increase understanding of adolescent determinants of breast density and identify physiological mechanisms underlying the association of adiposity and dietary fat intake in youth with adult breast density and possibly breast cancer risk. Metabolites identified could potentially be targeted as part of a novel preventive strategy.